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Journal of Food Protection, Vol. 79, No. 9, 2016, Pages 1623–1629
doi:10.4315/0362-028X.JFP-16-070
Research Note
Rapid Identification of Shiga Toxin–Producing Escherichia coli
O Serogroups from Fresh Produce and Raw Milk Enrichment
Cultures by Luminex Bead–Based Suspension Array
JULIE A. KASE,
1
*ANNA MAOUNOUNEN-LAASRI,
1,2
AND ANDREW LIN
3
1
U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Division of Microbiology, College Park, Maryland 20740;
2
Oak Ridge
Research Institute for Science and Education, Oak Ridge, Tennessee 37830; and
3
U.S. Food and Drug Administration, Office of Regulatory Affairs, San
Francisco Laboratory, Alameda, California 94502, USA
MS 16-070: Received 12 February 2016/Accepted 14 May 2016
ABSTRACT
The U.S. Food and Drug Administration’s Bacteriological Analytical Manual (BAM) Chapter 4a describes a Luminex
microbead–based suspension array used to screen colonies for 11 clinically relevant Shiga toxin–producing Escherichia coli
(STEC) serogroups: O26, O45, O91, O103, O104, O111, O113, O121, O128, O145, and O157. We evaluated the usefulness of
this method to identify STEC-positive enrichment samples before agar plating. Twelve E. coli strains were added to three types of
fresh produce (bagged baby spinach, alfalfa sprouts, and cilantro) at levels near the detection limit of the test. A subset of these
strains (six O serogroups) was similarly evaluated in raw milk. For comparison, portions of each of the 168 enrichment cultures
were analyzed for serogroup by a real-time PCR assay and a Bio-Plex 200 assay with the bead-based suspensions. No false-
positive results were obtained. Of the 112 samples with a reported cycle threshold (C
T
) value, 101 undiluted, diluted, or extracted
enrichment cultures also produced ratios above 5.0 in the Bio-Plex assay. When PCR C
T
values approached or were greater than
35, Bio-Plex detection became less reliable. Using undiluted or extracted enrichment cultures resulted in a significantly larger
number of positive results. With the same enrichment material prepared for real-time PCR analysis as described in the BAM
Chapter 4a, the STEC microbead-based suspension array can accurately screen food enrichment cultures.
Key words: Bio-Plex 200 instrumentation; Food enrichment; Luminex; O serogroup identification; Shiga toxin–producing
Escherichia coli
Shiga toxin–producing Escherichia coli (STEC) strains
are a significant public health concern, causing an estimated
170,000 illnesses in the United States each year (14). Over
100 different E. coli O serogroups are associated with Shiga
toxin production, although not all STEC strains are
pathogenic for humans (1, 3). However, some STEC strains
are as virulent as O157:H7, causing bloody diarrhea, life
threating conditions such as hemorrhagic colitis (HC) and
hemolytic uremic syndrome (HUS), and even death (2, 7,
10). Relatively few cells of these pathogens are needed to
cause disease, and the consumption of contaminated foods
or drinking water or close contact with STEC-infected
animals can be important transmission routes for STEC
infections in humans (4, 5, 15).
Governmental food regulatory agencies struggle with
identifying pathogenic STEC so that contaminated material
can be removed from the food supply and isolates obtained
from clinical, food, and environmental samples can be
matched. Part of the challenge for regulatory agencies is
differentiating between pathogenic STECs and those that do
not have the potential to cause illness. Although certain
virulence factors, such as intimin, have been implicated as
indicators of pathogenic potential (13), certain STEC O
serogroups are associated with clinical disease more often
than other serogroups. For example, particular serogroups
are known to cause HC and HUS (O26, O103, O111, O121,
and O145), and O45 is associated with HC (3, 8); however,
other serogroups (O91, O113, and O128) may cause HC and
HUS but are less commonly isolated (1, 8).Other
serogroups also may emerge as public health concerns, as
occurred in 2011 with the E. coli strain O104:H4. A single
multinational E. coli O104:H4 infection outbreak resulted in
908 cases of HUS and 50 deaths in 16 countries, including
the United States (16).
The current version of the U.S. Food and Drug
Administration (FDA) Bacteriological Analytical Manual
(BAM) (6) contains a culture method for the recovery and
detection of O157:H7 and non-O157 STEC from leafy
produce, which is based on the enrichment of produce rinses
followed by the use of chromogenic and selective or
differential plating agars. Milk and other liquids are first
centrifuged, and then samples are enriched and plated on
those same agars. For detection of Shiga toxin (i.e., coded by
genes stx
1
and stx
2
), a small portion of the enrichment
material or suspect colonies from the plating agars are
* Author for correspondence. Tel: 240-402-2923; Fax: 301-436-
2915; E-mail: julie.kase@fda.hhs.gov.
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subjected to a real-time PCR assay. Colonies can be further
evaluated on a Bio-Plex 200 instrument using a Luminex
microsphere–based suspension array that identifies the O
serogroup of pure culture isolates of the 11 most clinically
relevant STEC serogroups: O26, O45, O91, O103, O104,
O111, O113, O121, O128, O145, and O157 (11). This
validated assay replaces the traditional serotyping method of
testing agglutination with O serogroup–specific antisera and
offers many advantages, including better accuracy and
higher throughput. Although this assay has been very
effective for confirming colonies picked from agar plates,
several days may pass until a colony is obtained, costing
valuable time. Use of this assay to screen enrichment
samples, as previous work has demonstrated with bacterial
colonies, would allow early indication of a positive sample
and the particular serogroup involved, which might better
direct the use of particular agars (9, 11). Following the FDA
BAM procedure (Chapter 4a, Section N) (6), we evaluated
the suitability of the Luminex microsphere–based suspen-
sion array for identifying STEC-positive test portions of
enrichment cultures of artificially contaminated fresh
produce (bagged baby spinach, alfalfa sprouts, and cilantro)
and raw cow’s milk.
MATERIALS AND METHODS
Source of fresh produce, raw milk, and inocula. Bagged
baby spinach, packages of alfalfa sprouts, and bunches of cilantro
were purchased from local supermarkets in the Washington, DC
metropolitan area. Raw milk was obtained from cows maintained
at the FDA Center for Veterinary Medicine (courtesy of Dr. Oscar
Chiesa) for research purposes. E. coli strains used as inocula were
O26:H11 (TW02295), O45:NM (TW01589), O91:(TW05662),
O103:H6 (TW04162), O111:NM (TW06315), O113:H21
(TW00125), O121:NM (TW08004), and O145:NM (TW07596)
from the Thomas S. Whittam STEC Center (Michigan State
University, East Lansing); O104:H4 from Lawrence Connolly
(Massachusetts Department of Public Health, Boston); O104:H21
from Dr. Peter Feng (FDA, Silver Spring, MD); and O128
(BM2020) from Dr. Robert Mandrell (U.S. Department of
Agriculture, Agricultural Research Service, Washington, DC).
Preparation of inocula, food inoculation, enrichment
procedure, and real-time PCR screening. Preparation of inocula,
food inoculation, enrichment procedure, and real-time PCR
screening were all completed using food enrichment cultures
produced in a previous study (9). Test portions of approximately
100 g of baby spinach, cilantro, or alfalfa sprouts or 800 ml of raw
milk were inoculated with E. coli strains, placed in a sealable
container, mixed thoroughly by shaking for several minutes or by
inverting the container three to five times (raw milk), held for 48 h
at 48C for aging, and then split into four 25-g test portions by
produce type or four 200-ml test portions (raw milk). Inoculum
levels of approximately 0.05 CFU/g of produce or 0.05 CFU/ml of
milk (or levels that would provide fractionally positive results as
empirically determined) were used. Fractionally positive results are
results where some of the test portions per experiment (ideally
50%) produce positive results and some produce negative results.
Enrichment cultures were grown in 13modified buffered peptone
water with pyruvate under the exact conditions described in the
BAM (6). Uninoculated portions of produce or raw milk were also
processed with the inoculated test portions and enriched, as
described above, as controls for cross-contamination during
processing of the inoculated material. Nucleic acid extraction from
1.0-ml enrichment culture samples was performed using the
DNeasy blood and tissue kit (Qiagen, Valencia, CA) according to
the manufacturer’s instructions for gram-negative bacteria.
Luminex microsphere–based suspension array and oper-
ation of the Bio-Plex 200 instrument. Primers and probes used
and the details of the PCR, bead hybridization, and Bio-Plex
analysis are described elsewhere (11). PCRs were conducted using
a C1000 thermocycler (Bio-Rad, Hercules, CA). The STEC
molecular serotyping protocol is provided in the BAM. Protocol
procedures were followed without deviation as described. The
resulting PCR product for each enrichment sample was run in
triplicate on the Bio-Plex 200 instrument, and mean results are
reported in Table 1. In some cases, PCR products were stored
overnight at 48C.
Statistical analysis. The number of positive Bio-Plex ratios
(Table 1) achieved using diluted, undiluted, and extracted
enrichment material was analyzed with Fisher’s exact test using
GraphPad software (www.graphpad.com). Significance was as-
sessed with a two-tailed Pvalue of less than 0.0001.
RESULTS
Eleven E. coli O serogroups (12 E. coli strains) were
evaluated in food enrichment cultures prepared with three
types of fresh produce: bagged baby spinach, alfalfa sprouts,
and cilantro. A smaller subset of six of these O serogroups
was similarly evaluated in raw cow’s milk. For comparison,
portions of each of the 168 enrichment cultures were
analyzed by both real-time PCR and the Luminex
microsphere–based suspension array on the Bio-Plex 200
instrument. Through the use of a dual laser system, the Bio-
Plex 200 system can measure and quantitate fluorescence
signals for each microsphere detected and can categorize the
microsphere by region. Data are expressed as median
fluorescent intensities for each microsphere region. For each
sample, a signal-to-background ratio is calculated by
dividing the median fluorescent intensity for a particular
bead region by the corresponding intensity generated by a
no-template control sample. Based on the assay validation,
when this ratio exceeds 5.0, the sample is considered
positive for that serogroup (11, 12).
Real-time PCR analysis was conducted once for each
serotype and food commodity following instructions in
Section O of Chapter 4a in the BAM (6). Table 1 provides
results of both the real-time PCR and Bio-Plex assays for all
E. coli strains tested in the four food matrices. For the real-
time PCR assay, cycle threshold (C
T
) values are reported
and are compared with the ratios generated for the same
sample with the Bio-Plex 200 instrument. For the 168
samples analyzed, no false-positive results were obtained,
i.e., a sample negative for E. coli with the real-time PCR
assay was also negative with the Bio-Plex assay. Results for
the O128 serogroup are not shown in Table 1 because no
positive enrichment samples were obtained for any test
portions by real-time PCR or the Bio-Plex assay. Consul-
tation with the provider revealed that this particular O128
strain does not possess either of the Shiga toxin genes (stx
1
or stx
2
) detected by the BAM real-time PCR method,
making it difficult to compare results with those of the Bio-
1624 KASE ET AL. J. Food Prot., Vol. 79, No. 9
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TABLE 1. Results of analysis of enrichment culture samples analyzed by both the Bio-Plex 200 instrument and real-time PCR
a
E. coli
serogroup Trial
Baby spinach Cilantro Alfalfa sprouts Raw milk
b
Bio-Plex PCR (C
T
) Bio-Plex PCR (C
T
) Bio-Plex PCR (C
T
) Bio-Plex PCR (C
T
)
O26 1 ,5.0 Pos (27.03 stx
1
),5.0 ND Pos Pos (26.80 stx
1
),5.0 Pos (39.21 stx
1
)
Pos
c
,5.0
c
Pos
d
,5.0
d
2,5.0 ND ,5.0 Pos (39.07 stx
1
) Pos Pos (34.35 stx
1
),5.0 Pos (35.64 stx
1
)
,5.0
c
Pos
c
,5.0
d
Pos
d
3,5.0 ND Pos Pos (24.49 stx
1
) Pos Pos (26.85 stx
1
),5.0 Pos (38.59 stx
1
)
,5.0
c
,5.0
d
4,5.0 ND ,5.0 Pos (39.20 stx
1
) Pos Pos (26.45 stx
1
),5.0 Pos (38.29 stx
1
)
,5.0
c
,5.0
c
,5.0
d
,5.0
d
O45 1 ,5.0 ND Pos Pos (24.25 stx
1
),5.0 ND ,5.0 ND
2 Pos Pos (24.59 stx
1
),5.0 Pos (37.75 stx
1
),5.0 ND ,5.0 ND
Pos
c
Pos
d
3 Pos Pos (23.54 stx
1
),5.0 ND ,5.0 ND ,5.0 ND
4 Pos Pos (24.08 stx
1
) Pos Pos (24.99 stx
1
),5.0 ND ,5.0 ND
O91 1 Pos Pos (25.48 stx
1
),5.0 ND Pos Pos (30.76 stx
1
)
2 Pos Pos (26.15 stx
1
) Pos Pos (26.50 stx
1
) Pos Pos (31.36 stx
1
)
3 Pos Pos (26.87 stx
1
),5.0 ND ,5.0 ND
4 Pos Pos (29.34 stx
1
) Pos Pos (26.09 stx
1
) Pos Pos (32.31 stx
1
)
O103 1 Pos Pos (23.09 stx
1
) Pos Pos (23.10 stx
1
),5.0 ND Pos Pos (30.08 stx
1
)
2,5.0 ND Pos Pos (22.40 stx
1
) Pos Pos (26.76 stx
1
) Pos Pos (29.52 stx
1
)
3 Pos Pos (23.28 stx
1
) Pos Pos (22.69 stx
1
) Pos Pos (25.50 stx
1
) Pos Pos (30.36 stx
1
)
4,5.0 ND ,5.0 ND Pos Pos (25.01 stx
1
) Pos Pos (30.71 stx
1
)
O104:H4 1 Pos Pos (25.86 stx
2
) Pos Pos (27.81 stx
2
) Pos Pos (31.25 stx
2
)
2,5.0 Pos (35.57 stx
2
) Pos Pos (30.26 stx
2
) Pos Pos (33.31 stx
2
)
Pos
c
Pos
d
3 Pos Pos (27.14 stx
2
) Pos Pos (29.20 stx
2
) Pos Pos (33.46 stx
2
)
4 Pos Pos (26.07 stx
2
) Pos Pos (28.41 stx
2
) Pos Pos (32.04 stx
2
)
O104:H21 1 ,5.0 Pos (36.63 stx
2
) Pos Pos (27.08 stx
2
),5.0 Pos (38.33 stx
2
),5.0 Pos (35.36 stx
2
)
Pos
c
Pos
c
Pos
c
Pos
d
Pos
d
Pos
d
2,5.0 Pos (36.12 stx
2
) Pos Pos (26.80 stx
2
),5.0 Pos (35.09 stx
2
),5.0 ND
Pos
c
Pos
c
Pos
d
Pos
d
J. Food Prot., Vol. 79, No. 9 E. COLI O SEROGROUP IDENTIFICATION FROM FOOD ENRICHMENTS 1625
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TABLE 1. Continued
E. coli
serogroup Trial
Baby spinach Cilantro Alfalfa sprouts Raw milk
b
Bio-Plex PCR (C
T
) Bio-Plex PCR (C
T
) Bio-Plex PCR (C
T
) Bio-Plex PCR (C
T
)
O104:H21
(cont’d)
3 Pos Pos (23.98 stx
2
) Pos Pos (35.10 stx
2
),5.0 Pos (36.82 stx
2
) Pos Pos (36.05 stx
2
)
Pos
c
Pos
d
4 Pos Pos (26.22 stx
2
) Pos Pos (33.93 stx
2
),5.0 Pos (36.63 stx
2
) Pos Pos (32.04 stx
2
)
Pos
c
Pos
d
O111 1 ,5.0 ND ,5.0 ND ,5.0 ND ,5.0 Pos (37.62 stx
1
; 39.05 stx
2
)
,5.0
c
,5.0
d
2,5.0 ND ,5.0 Pos (35.47 stx
1
; 37.96 stx
2
),5.0 Pos (39.24 stx
1
; 36.60 stx
2
),5.0 Pos (39.44 stx
1
; 38.75 stx
2
)
,5.0
c
,5.0
c
,5.0
c
Pos
d
(7.13) ,5.0
d
,5.0
d
3,5.0 ND ,5.0 Pos (35.24 stx
1
; 37.54 stx
2
),5.0 Pos (stx
1
ND; 38.64 stx
2
),5.0 ND
,5.0
c
Pos
c
Pos
d
(7.37) Pos
d
4,5.0 ND ,5.0 Pos (37.15 stx
1
; 38.99 stx
2
),5.0 Pos (stx
1
ND; 38.52 stx
2
),5.0 Pos (36.62 stx
1
; 36.64 stx
2
)
,5.0
c
,5.0
c
,5.0
c
,5.0
d
,5.0
d
,5.0
d
O113 1 ,5.0 ND ,5.0 ND ,5.0 Pos (37.94 stx
2
)
Pos
c
Pos
d
2 Pos Pos (28.50 stx
2
) Pos Pos (29.25 stx
2
),5.0 Pos (39.26 stx
2
)
Pos
c
Pos
d
3 Pos Pos (28.62 stx
2
),5.0 ND ,5.0 Pos (36.11 stx
2
)
Pos
c
Pos
d
4,5.0 ND Pos Pos (29.36 stx
2
),5.0 ND
O121 1 ,5.0 ND Pos Pos (25.22 stx
2
),5.0 ND
2 Pos Pos (23.53 stx
2
) Pos Pos (26.19 stx
2
),5.0 Pos (39.78 stx
2
)
Pos
c
Pos
d
3 Pos Pos (24.09 stx
2
),5.0 ND ,5.0 Pos (36.24 stx
2
)
Pos
c
Pos
d
4,5.0 ND ,5.0 ND ,5.0 Pos (38.51 stx
2
)
Pos
c
Pos
d
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TABLE 1. Continued
E. coli
serogroup Trial
Baby spinach Cilantro Alfalfa sprouts Raw milk
b
Bio-Plex PCR (C
T
) Bio-Plex PCR (C
T
) Bio-Plex PCR (C
T
) Bio-Plex PCR (C
T
)
O145 1 ,5.0 Pos (38.66 stx
1
) Pos Pos (22.33 stx
1
),5.0 ND ,5.0 ND
Pos
c
Pos
d
2 Pos Pos (21.78 stx
1
),5.0 ND ,5.0 ND ,5.0 Pos (34.59 stx
1
)
Pos
c
Pos
d
3 Pos Pos (21.35 stx
1
) Pos Pos (33.60 stx
1
),5.0 ND ,5.0 Pos (37.19 stx
1
)
Pos
c
Pos
d
4 Pos Pos (21.32 stx
1
) Pos Pos (22.96 stx
1
) Pos Pos (21.13 stx
1
),5.0 Pos (37.12 stx
1
)
Pos
c
Pos
d
O157:H7 1 ,5.0 Pos (38.90 stx
1
; 37.33 stx
2
) Pos Pos (32.60 stx
1
; 31.33 stx
2
),5.0 Pos (38.45 stx
1
;stx
2
ND)
Pos
c
,5.0
c
Pos
d
Pos (20.1)
d
2 Pos Pos (26.99 stx
1
; 25.29 stx
2
) Pos Pos (32.53 stx
1
; 31.37 stx
2
),5.0 Pos (stx
1
ND; 37.78 stx
2
)
Pos
c
Pos
d
3 Pos Pos (25.12 stx
1
; 23.50 stx
2
) Pos Pos (32.97 stx
1
; 31.42 stx
2
),5.0 Pos (stx
1
ND; 38.81 stx
2
)
Pos
c
Pos
d
4,5.0 Pos (stx
1
ND; 37.89 stx
2
) Pos Pos (34.67 stx
1
; 32.41 stx
2
),5.0 Pos (38.27 stx
1
; 37.55 stx
2
)
Pos
c
Pos
c
Pos
d
Pos
d
a
Signal-to-background ratios are reported for the Bio-Plex assay, and C
T
values are given for the real-time PCR assay. The resulting PCR product for each enrichment culture sample was run in
triplicate on the Bio-Plex 200 instrument, and mean ratios are reported. Bold results indicate a possible limit of detection for the Bio-Plex 200 assay when testing fresh produce food enrichment
cultures. When a test portion produced a C
T
value but did not generate a Bio-Plex ratio above 5.0, the undiluted and extracted enrichment material was tested. Results for O128 serogroup are not
shown because no positive results were obtained for any enrichment culture test portions. ND, not detected (no C
T
value generated).
b
Not all E. coli serogroups were evaluated in raw milk.
c
Result for undiluted enrichment culture.
d
Result for extracted enrichment culture.
J. Food Prot., Vol. 79, No. 9 E. COLI O SEROGROUP IDENTIFICATION FROM FOOD ENRICHMENTS 1627
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Plex assay. All uninoculated (control) portions processed
with the corresponding inoculated samples were negative
with both assays (data not shown). All positive and negative
(no template) controls run with these samples also
performed as expected. Of the 112 diluted samples with a
reported C
T
value, 71 also produced ratios above 5.0 in the
Bio-Plex assay (Table 1). A closer look at those diluted test
portions that produced a negative result in the Bio-Plex
assay (shown in bold in Table 1) revealed that the
corresponding C
T
values were close to or greater than 35.
This was true for 41 test portions across all four types of
food, with three notable exceptions: baby spinach O26 trial
1, cilantro O104:H21 trial 3, and raw milk O104:H21 trial 3.
For the diluted cilantro enrichment, a strong ratio of 19.58
was obtained for the Bio-Plex assay, and the corresponding
real-time PCR C
T
was 35.10. However, in the raw milk trial,
the Bio-Plex ratio was 5.34, close to the 5.0 cutoff for a
positive result and the C
T
was higher, 36.05. For baby
spinach O26 trail 1, the C
T
was 27.03, but the parallel Bio-
Plex assay result was below 5.0. However, use of both the
undiluted and extracted enrichment material produced a
positive Bio-Plex result. These three findings suggest that a
real-time PCR C
T
of 35 might not be an absolute breaking
point in terms of obtaining a positive Bio-Plex assay result.
When a test portion produced a C
T
value but did not
generate a Bio-Plex ratio above 5.0, the undiluted and
extracted enrichment material was tested (Table 1). Overall,
this additional testing resulted in a significant increase (P,
0.0001) in positive test portions for undiluted (increase of
27) or extracted (increase of 30) material. DNA extraction
resulted in positive samples for O111 cilantro trials 2 and 3
and O157:H7 alfalfa sprouts trial 1. However, the Bio-Plex
ratios for the cilantro trials were very close to the cutoff of
5.0 (7.1 and 7.4, respectively). For O157:H7 alfalfa sprouts
trial 1, the resulting ratio was 20.1.
DISCUSSION
The current BAM (6) describes characterization of
STEC isolates by detecting virulence factors (e.g., stx
1
or
stx
2
) and identifying the most clinically relevant O
serogroups using a Luminex microsphere–based suspension
array. The array can identify the 11 most clinically relevant
STEC serogroups (O26, O45, O91, O103, O104, O111,
O113, O121, O128, O145, and O157) and is performed in
96-well plates, allowing for high-throughput screening for a
large number of analytes. Previous studies have demon-
strated that the assay is reproducible (giving positive results
more than two standard deviations above the threshold
value), accurate (identifying 114 STECs correctly with no
false-positive results among 46 negative control isolates),
and robust (correctly identifying STECs with 96.4 to 100%
accuracy in a blinded collaborative study involving 55
unknown strains and nine laboratories) (11, 12).The
Luminex array also is adaptable, producing accurate results
on both the Bio-Plex 200 and MAGPIX systems with only
minor adjustments.
Because this assay has demonstrated its usefulness for
confirming certain STEC colonies, we decided to evaluate
its use with early positive enrichment samples before
incubation on agar plates. This adaptation requires no
additional effort or modification because the same enrich-
ment aliquot is prepared for real-time PCR analysis and the
same Bio-Plex reagents and procedures currently outlined in
the BAM can be used. Often the process of screening
colonies from multiple agars is laborious and expensive for
samples ultimately deemed negative for pathogenic E. coli.
Worse is the potential to miss positive colonies because only
subsets of colonies with typical characteristics are screened.
Initial screening of enrichment cultures for the presence of
certain pathogenic STEC would provide early and valuable
indication of a positive sample, which could result in a more
focused effort to obtain a colony isolate and possibly quicker
intervention to protect public health. For example, in a
previous study the agars recommended in the BAM varied in
performance depending on the E. coli O serogroup tested
(9). The enrichment culture screening method detailed here
would provide an early indication of the presence of certain
E. coli serogroups, and selection of a high-performance agar
would be possible based on results from the screening.
Screening would also provide information about typical
colony appearance for the agars evaluated, thus allowing
better use of limited colony picks. More aggressive attempts
might be made to obtain a positive colony from a food
sample culture with an early indication of a clinically
relevant E. coli strain.
This Luminex assay can be extended to effectively
screen food enrichment samples in addition to bacterial
colonies. However, care should be taken with regard to
negative Bio-Plex results. We verified that the sensitivity of
the conventional PCR step is less than that of the equivalent
real-time PCR analysis as indicated by Bio-Plex ratios below
5.0 when C
T
values approach 35. This limitation was
expected because generally smaller amounts of DNA are
detectable with the real-time PCR assay. For this study, we
attempted to obtain a mix of positive and negative results for
thorough assay evaluation and subsequently obtained C
T
values close to the maximum of 40. For 41 samples a C
T
value was recorded in the absence of a positive Bio-Plex
assay result (Table 1); therefore, a Bio-Plex ratio of less than
5.0 does not always mean that the sample does not contain
an STEC belonging to one of the 11 serogroups that are part
of the Bio-Plex assay. However, in a natural contamination
event and with subsequent robust food enrichment, the Bio-
Plex assay should be able to detect a positive sample,
because more DNA template should be present. Analysis of
undiluted enrichment cultures resulted in a 24% increase in
the number of positive samples. However, as with any
molecular assay, positive signals can be generated from both
live and dead bacterial cells.
In summary, with no modification to the current Bio-
Plex assay method detailed in Chapter 4a of the BAM and
using the same enrichment material prepared for real-time
PCR analysis, the STEC microbead-based suspension array
can be used to accurately screen food enrichments for the 11
O serogroups validated for the current assay. Thus, we
recommend the expanded use of this assay as a rapid first
screen of undiluted E. coli food enrichment cultures to
provide an early indication of contamination with potentially
pathogenic STEC. Rapid and high-throughput identification
and serotyping of STEC O serogroups is important for
1628 KASE ET AL. J. Food Prot., Vol. 79, No. 9
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detecting, investigating, and controlling STEC infection
outbreaks and removing hazardous products from com-
merce.
ACKNOWLEDGMENT
This project was supported in part by an appointment (A.
Maounounen-Laasri) to the Research Participation Program at the Center
for Food Safety and Applied Nutrition administered by the Oak Ridge
Institute for Science and Education.
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